WO2021055968A1 - Procédés d'évaluation de la compatibilité d'une greffe pour la transplantation - Google Patents

Procédés d'évaluation de la compatibilité d'une greffe pour la transplantation Download PDF

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WO2021055968A1
WO2021055968A1 PCT/US2020/051847 US2020051847W WO2021055968A1 WO 2021055968 A1 WO2021055968 A1 WO 2021055968A1 US 2020051847 W US2020051847 W US 2020051847W WO 2021055968 A1 WO2021055968 A1 WO 2021055968A1
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graft
dna
amount
fragment
cell
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PCT/US2020/051847
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English (en)
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Paula E. North
Michael E. MITCHELL
Aoy T. MITCHELL
Paul G. DAFT
Donna K. MAHNKE
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Natera Inc.
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Priority to US17/761,311 priority Critical patent/US20220340963A1/en
Publication of WO2021055968A1 publication Critical patent/WO2021055968A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Definitions

  • compositions and methods for assessing graft suitability for transplantation are provided herein.
  • methods for assessing graft suitability for transplantation by measuring total and/or graft- specific cell-free nucleic acids, such as cell- free DNA.
  • transplantation rejection is an impediment to long-term transplantation success.
  • Acute transplantation rejection can occur within days to weeks after transplantation and may lead to chronic rejection and gradual loss of function of the transplanted organ.
  • rejection remains a major complication post-transplantation. Accordingly, methods for identifying grafts that are suitable for transplantation are needed.
  • compositions and methods for assessing graft suitability for transplantation are provided herein.
  • a method of assessing the suitability of a potential graft for transplantation may be done by identifying the absolute or steady- state level of cell-free DNA.
  • Total cell free DNA of a graft ex vivo in a perfusion container may include contents indicative of lysis and/or apoptosis of cells from the graft and from blood from the donor. As a graft starts to deteriorate, apoptosis of cells may increase and total cell free DNA levels may also increase.
  • the levels can be evaluated by comparison to a threshold of cellular or blood components, for example a threshold level of cfDNA.
  • a suitable graft for transplant would preferably have low or steady-state levels of total cell free DNA.
  • the method comprises obtaining an amount of total short fragment cf-DNA and/or graft- specific short-fragment cf-DNA released from a graft and assessing the amount(s) to determine the suitability of the graft for transplantation.
  • the amount may be obtained prior to contacting the graft or cells thereof with blood cells from a potential recipient and/or subsequent to contacting the graft or cells thereof with blood cells from a potential recipient.
  • obtaining an amount comprises performing a method to quantify the amount of total short fragment cf-DNA and/or graft- specific short fragment cf-DNA released by the graft.
  • the methods further comprise obtaining an amount of cell-lysis, such as by performing fragment analysis.
  • the graft is monitored over time.
  • the methods described herein may involve obtaining an amount of total short fragment cf-DNA and/or graft- specific short-fragment cf-DNA and/or determining the amount of cell lysis at one or more additional time points.
  • the method may further comprise comparing the amount(s) of the total short fragment cf-DNA and/or graft- specific cf-DNA and/or amount of cell lysis with threshold value(s) or amount(s) obtained from the additional time point(s).
  • the amount(s) of the total short fragment cf-DNA, graft- specific cf-DNA, and/or cell lysis is measured using a quantitative amplification method. In other aspects, the amount(s) is measured using next-generation sequencing.
  • the amount(s) of the total short fragment cf-DNA and/or graft- specific cf-DNA and/or amount of cell lysis are obtained in a sample of media in which the potential graft or cells therefrom are contained or are in contact with.
  • the media may be a storage, perfusion, or preservation media in which the graft is contained or in contact with.
  • the graft is an organ or organs.
  • the graft may be a heart, a lung, or a heart and a lung.
  • the graft may be from a donor of the same species as the potential recipient.
  • the method may further comprise providing the values for the amount(s) of total cell-free DNA and/or graft- specific cf-DNA and/or cell lysis in a report.
  • the report may be used by clinicians to assess the suitability of the graft for transplantation.
  • the graft is transplanted into a subject if the graft is identified as suitable for transplantation.
  • FIG. 1 shows reference gene qPCR measuring the percentage of detectable DNA after spiking a known quantity of sheared gDNA into STEEN solutionTM containing heparin or no heparin.
  • FIG. 2 shows reference gene qPCR measuring the percentage of detectable DNA after spiking 20 ng of sheared gDNA into STEEN SolutionTM supplemented with heparin at 0, 0.984, 1.125, and 1.5 IU/ml.
  • FIG. 3 shows reference gene qPCR measuring the percentage of detectable DNA after spiking 500 ng/ml of sheared gDNA into STEEN SolutionTM containing 0, 1, 5, or 100 IU/ml of heparin.
  • FIG. 4 shows reference gene qPCR measuring the percent recovery of a short fragment DNA control from STEEN SolutionTM containing 0, 1, 5, or 100 IU/ml of heparin.
  • FIG. 5 shows reference gene qPCR measuring the DNA concentration of purified and concentrated spiked STEEN solutionTM samples vs. unpurified 0.1X TE controls at concentrations of 500 and 1000 ng/ml.
  • FIG. 6 shows reference gene qPCR measuring the DNA concentration of purified and concentrated spiked STEEN solutionTM samples vs unpurified 0.1X TE controls at concentrations of 10 and 40 ng/ml.
  • FIG. 7 shows reference gene qPCR measuring DNA concentration in STEEN SolutionTM and plasma after 0, 2, 4, and 6-hour incubations at 37°C.
  • FIG. 8 shows reference gene qPCR measuring DNA concentrations in STEEN solutionTM after incubation at 22°C (RT) or 37°C for 0, 1, 3, 12, and 24 hours.
  • FIG. 9 shows reference gene qPCR measuring DNA concentrations in plasma after incubation at 22°C (RT) or 37°C for 0, 1, 3, 12, and 24 hours.
  • Embodiments of the present disclosure provide compositions and methods for assessing graft suitability for transplantation.
  • the present disclosure provides compositions and methods for assessing cell-free nucleic acids in a graft and assessing suitability of the graft for transplantation based upon these measurements.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, llamas, camels, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, rabbits, guinea pigs, and the like. The subject may be any age or sex.
  • Total cell free DNA of a graft ex vivo in a perfusion container generally will represent lysis and/or apoptosis of cells from the graft and any cells from blood from the donor. Without being bound by theory, it is thought that as a graft starts to deteriorate, apoptosis of cells increases, and total cell free DNA levels also increases.
  • a suitable graft for transplant generally has low or steady-state levels of total cell free DNA.
  • the present disclosure provides methods for assessing graft suitability for transplantation.
  • the methods comprise obtaining an amount of cell-free nucleic acids (such as cell-free DNA) released from a graft and assessing the amount to determine the suitability of the potential graft for transplantation.
  • the cell-free nucleic acids are short-fragment (e.g. less than or equal to 170bps) cell-free DNA.
  • the methods comprise obtaining an amount of short fragment (e.g. fragments less than or equal to 170 bps) cell-free DNA released from a graft. The amount of short fragment cell- free DNA is then used to assess suitability of the potential graft for transplantation.
  • the methods provided herein or otherwise known in the art can be used multiple times to obtain total and/or specific cell-free nucleic acid (such as DNA) values over time. Also included herein are reports that include one or more of these values. Such reports provide valuable information to a clinician. In some embodiments, the clinician then assesses the condition (or suitability of a graft) and/or makes treatment decisions accordingly for a subject.
  • nucleic acid such as DNA
  • graft refers to a biological material comprising cells or tissue, such as at least a portion of an organ, that may be transplanted or implanted in or into a subject.
  • the graft is explanted material comprising cells or tissue, such as at least a portion of an organ that is being maintained outside the body (ex vivo), such as to preserve or rehabilitate, the graft.
  • Organs and tissues include, but are not limited to, heart, kidney, liver, lung, pancreas, intestine, thymus, bone, tendon, cornea, skin, heart valve, nerve, and blood vessel (e.g., vein).
  • the graft is a whole organ or more than one organ.
  • organs that can be transplanted or implanted include, but are not limited to, the heart, kidney(s), kidney, liver, lung(s), pancreas, intestine, etc.
  • the graft is more than one organ.
  • the graft may be a combination of a heart and lung.
  • the graft is one organ.
  • the graft may be a lung.
  • the graft a portion of an organ.
  • the graft may be a valve.
  • Grafts may be of the same species as the potential recipient of the graft, or may be of a different species from the potential recipient of the graft.
  • the graft is from a different species (i.e., is a xenograft) than the recipient.
  • the graft may be from a pig or cow and the recipient may be a human. Any one of the types of grafts provided herein may be a xenograft.
  • the graft is a pig or cow valve.
  • the graft is from the same species as the potential recipient of the graft.
  • the graft may be from a human and the recipient may be a different human.
  • the graft is decellularized graft, such as a decellularized xenograft.
  • the graft is an autograft. Any one of the methods or compositions provided herein may be used for assessing any one of the grafts described herein. Any one of the methods provided herein can be used to evaluate suitability for future engraftment. For example, the methods provided herein can be used to evaluate suitability of a graft isolated from one subject for suitability for engraftment into another subject.
  • the methods described herein comprise obtaining a value of cell-free DNA from a sample obtained from or in contact with the graft.
  • the methods comprise obtaining a value of short fragment cell-free DNA from a sample obtained from or in contact with the graft.
  • the sample can be a biological sample. Examples of such biological samples include whole blood, plasma, serum, etc.
  • the sample comprises media in which the graft is placed or with which it has contact.
  • the media comprises blood or a blood substitute, preservation solution, or any other solution in which a graft is placed or with which it has contact, such as in in vitro contexts.
  • the graft such as an organ or organs, is contained in a perfusion system.
  • the one or more samples and/or one or more other samples are obtained within minutes, such as no more than 15, 20, 25, 30, 35, 40, 45, 50, or 55 minutes, of obtaining the graft (e.g., storing the graft, perfusing the graft, etc.).
  • the one or more samples and/or one or more other samples are obtained within hours, such as no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18 or more hours, of obtaining the graft (e.g., storing the graft, perfusing the graft, etc.)
  • more than one sample is obtained.
  • an initial sample may be obtained, and one or more additional samples may be obtained at subsequent time points.
  • the one or more other subsequent time points are at hourly intervals.
  • an initial sample is obtained within an hour of obtaining the graft and one or more other samples are obtained within 15, 20, 25, 30, 35, 40, 45, 50, or 55 minute intervals or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18 or more hourly intervals, such as until a threshold value or baseline is reached.
  • the one or more other subsequent time points are at daily intervals.
  • the one or more other subsequent time points are at one- week intervals. In some embodiments of any one of the methods provided herein, the one or more other subsequent time points are at two-week intervals. In some embodiments of any one of the methods provided herein, the one or more other subsequent time points are at monthly intervals.
  • the sample may be subjected to various methodologies prior to obtaining the level of cell-free nucleic acids in the sample. For example, the sample may be subjected to one or more centrifugation steps. In some embodiments, the sample is centrifuged once. In some embodiments, the sample is centrifuged more than once.
  • the sample may be centrifuged once, two times, three times, four times, five times, or more than five times prior to obtaining the value of cell-free DNA in the sample.
  • the centrifugation steps may be performed at any suitable speed for any suitable duration to achieve the desired result.
  • the sample may be centrifuged at 1000 x g, 1100 x g, 1200 x g, 1300 x g, or 1400 x g.
  • the sample is centrifuged at the desired speed within 2 hours of sample collection.
  • the sample is centrifuged once or multiple times at a first sample collection site and shipped to a second site for quantification of cell-free nucleic acids in the sample.
  • the sample may be centrifuged 2-3 times at 1100 x g, 1200 x g, 1300 x g, or 1400 x g within 2 hours of collection, frozen, and shipped to a second site for quantification of cell-free nucleic acids in the sample.
  • the graft e.g., cells, tissue, organ
  • graft storage media can be intracellular (e.g., perfused) or extracellular, and may depend on the graft to be preserved.
  • Approaches to preserving most grafts include simple static cold storage (SCS) and dynamic preservation. Examples of dynamic preservation include hypothermic machine perfusion (HMP), normothermic machine perfusion, and oxygen persufflation.
  • HMP hypothermic machine perfusion
  • normothermic machine perfusion normothermic machine perfusion
  • oxygen persufflation oxygen persufflation
  • the graft storage media can prevent clotting in harvests with blood present, reduce stress and deterioration associated with ex vivo handling, and decrease the risk of microbial growth. Therefore, in some embodiments, the graft storage media comprises osmotic active agents, electrolytes, hydrogen ion buffers, colloid(s), metabolic inhibitors, metabolites, and antioxidants. Examples of osmotic active agents, which may prevent cell swelling, include lactobionate, raffinose, citrate, and gluconate. Electrolytes, which can exert an osmotic effect, include sodium, potassium, calcium, and magnesium ions.
  • Examples of hydrogen ion buffers include phosphate, histidine, and N-(2-hydroxyethyl)-piperazine-N’-2-ethanesulfonic acid (HEPES) buffer.
  • colloids which may be used during the initial vascular flush out and perfusion, include albumin and HES.
  • examples of metabolic inhibitors which may suppress degradation of cell constituents, include allopurinol, antiproteases, and chlorpromazine.
  • Examples of metabolites which can help restore metabolism during the reperfusion phase, include adenosine, glutathione, and phosphate.
  • Examples of antioxidants, which can inhibit oxygen free-radical injury include steroids, vitamin E, deferoxamine, and tryptophan.
  • Graft storage media are commercially available, and examples include BELZER UW® cold storage solution (VIASPANTM or the University of Wisconsin (UW) solution), CELSIOR®, CUSTODIOL®, and IGL-1®.
  • the graft is a lung or lungs.
  • the compositions and methods provided herein may be used to determine suitability of donor lungs for transplantation.
  • the graft is one or more ex-vivo lung perfusion (EVLP) lungs.
  • the methods are applied to EVLP lungs that have been removed from one subject to evaluate suitability for engraftment into another subject. Accordingly, provided herein is a method for monitoring cellular injury in ex-vivo lung perfusion (EVLP) lungs during perfusion to assess suitability of the lung for transplantation.
  • EVLP ex-vivo lung perfusion
  • the methods described herein are used to evaluate the potential contribution of cf-DNA released from leukocytes or other cell types emanating from the perfused lungs, as well as potential interference with cf-DNA analysis by molecular additives to the EVLP system, such as heparin or dextran. In some embodiments, these preparatory determinations are used to provide accurate reporting of total cf-DNA in EVLP perfused lungs, and donor fraction in lung transplant patients.
  • STEENTM solution is a buffered extracellular solution that includes human serum albumin (HSA) to provide optimal osmotic pressure, facilitated by Dextran 40, a mild scavenger to coat and protect the endothelium from excessive leucocyte interaction.
  • HSA human serum albumin
  • EVLP using STEENTM solution thus has the potential to be able to increase the likelihood that previously rejected , but ex vivo rehabilitated lungs could be used to increase the availability of potential organs for lung transplantation. Accordingly, in some embodiments the sample is STEENTM solution that has been in contact with the graft.
  • the methods for assessing suitability of a graft for transplantation described herein comprise obtaining an amount of cell-free nucleic acids released from a graft and assessing the amount of cell-free nucleic acid to determine the suitability of the potential graft for transplantation.
  • the methods include obtaining a value for the amount of total cell-free nucleic acids (such as DNA) and/or a value for the amount of specific cell-free nucleic acids (such as DNA).
  • a “value” is any indicator that conveys information about an “amount.”
  • the indicator can be an absolute or relative value for the amount.
  • “amount” refers to the quantity of nucleic acids (such as DNA). Further, the value can be the amount, frequency, ratio, percentage, etc.
  • a “threshold value” refers to any predetermined level or range of levels that is indicative of a state, the presence or absence of a condition or the presence or absence of a risk.
  • the threshold value can take a variety of forms. It can be single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as where the risk in one defined group is double the risk in another defined group.
  • a threshold value is a baseline value, such as without the presence of a state, condition or risk or after a course of treatment or other remedial action.
  • specific cell-free nucleic acids refers to a subset of cell-free nucleic acids (such as DNA) that is within the total cell-free nucleic acids (such as DNA).
  • the specific cell-free nucleic acids are cell-free nucleic acids (such as DNA) that are graft- specific (GS).
  • GS cf-DNA refers to DNA that presumably is shed from the graft or cells thereof, the sequence of which matches (in whole or in part) the genotype of the subject from which the graft is obtained.
  • GS cf-DNA may refer to certain sequence(s) in the GS cf-DNA population, where the sequence is distinguishable from the recipient or potential recipient cf-DNA (e.g., having a different sequence at a particular nucleotide location(s)), or it may refer to the entire GS cf-DNA population).
  • the GS cf-DNA is short fragment (e.g. less than or equal to 170bp) GS cf-DNA.
  • the values for the amount(s) of nucleic acids can be “obtained” by any one of the methods provided herein or other methods known in the art, and any obtaining step(s) can include any one of the methods incorporated herein by reference or otherwise provided herein.
  • “Obtaining” as used herein refers to any method by which the respective information or materials are acquired.
  • the respective information e.g. the amount of cell-free DNA
  • Respective materials can be created, designed, etc. with various experimental or laboratory methods, in some embodiments.
  • the respective information or materials can also be acquired by being given or provided with the information, such as in a report, or materials.
  • the amount of cell-free DNA can be obtained by being provided with a report containing the value of the amount of cell-free DNA in a sample.
  • materials are given or provided through commercial means (i.e. by purchasing the materials from a third party service laboratory).
  • the suitability can be determined using one or more values for the amount of total cell-free nucleic acids (such as DNA) and/or one or more values for the amount of specific cell-free nucleic acids (such as DNA).
  • contaminating intact cells are removed from samples, such as perfusate samples, by one or more (e.g., one, two or three or more) centrifugation steps.
  • a baseline is established for meaningful cf-DNA analysis after effective washout of contaminating leukocytes.
  • the suitability can also be determined using one or more values for the amount of total cell-free nucleic acids (such as DNA) and/or one or more values from fragment analysis.
  • Cell lysis such as from mechanical stress and degenerative changes after sample collection, as opposed to apoptosis, releases long genomic fragments in biological samples, such as blood samples.
  • the majority of cf-DNA from blood drawn from normal healthy individuals appears to be the result of normal cellular apoptosis and turnover.
  • Apoptosis results in the release of relatively short DNA fragments, significantly shorter than those released by cellular lysis.
  • Samples in which significant cell lysis has occurred e.g. samples that contain higher proportions of long fragments
  • fragment analysis is performed to evaluate potential contaminating factors, such as excess long fragment DNA, that may negatively impact the ability to accurately quantify cf-DNA in the graft.
  • fragment analysis is performed by assessing short and/or long nucleic acid fragments.
  • a “long fragment” refers to a fragment that is greater than 170 bps (e.g., between 171 and 300 bps in length), while a “short fragment” is a fragment that is less than or equal to 170 bps.
  • a short fragment may be between 75 and 170 bps in length.
  • Such methods generally are performed with primers targeting a long fragment and/or a short fragment.
  • the fragment can be an Alu fragment.
  • An Alu element is a short stretch of DNA originally characterized by the action of the Arthrobacter luteus (Alu) restriction endonuclease. Alu repeats are the most abundant sequences in the human genome, with a copy number of about 1.4 million per genome. Alu sequences are short interspersed nucleotide elements (SINEs), typically 300 nucleotides, which account for more than 10% of the genome.
  • SINEs short interspersed nucleotide elements
  • a high proportion of long fragment DNA in the sample may negatively impact accurate cf-DNA measurements.
  • the methods comprise obtaining an amount or a value of short-fragment cell-free nucleic acids in the sample. For example, long DNA fragments are removed from the sample and the remaining short-fragment cell-free DNA are measured. Alternatively, short DNA fragments are isolated from the sample and short-fragment cell-free nucleic acids are subsequently measured. Any suitable materials and methods for removing long DNA fragments or isolating short DNA fragments in the sample may be used. Suitable materials and methods include, for example, size-based purification methods including gel electrophoresis, particles (e.g.
  • suitable methods include silica-based methods, (e.g. silica beads, paramagnetic silica particles, lysine- functionalized silica particles, PEG-modified silica particles, carboxyl-functionalized silica particles, lysine-functionalized silica particles, silica matrixes, amine-modified silica matrixes, metal ion-modified silica matrixes, etc.) to preferentially isolate short DNA and/or remove long-DNA from the sample.
  • silica-based methods e.g. silica beads, paramagnetic silica particles, lysine- functionalized silica particles, PEG-modified silica particles, carboxyl-functionalized silica particles, lysine-functionalized silica particles, silica matrixes, amine-modified silica matrixes, metal ion-modified silica matrixes, etc.
  • bead-based size-selective purification is performed to remove
  • long DNA fragments are removed by bead isolation, as described in Example 12.
  • the total cell-free nucleic acids and/or specific cell-free nucleic acids is quantified using short-fragment DNA in the sample. This value is used to assess suitability of the graft for transplantation.
  • the method further includes assessing the suitability (e.g., health, state, or condition) of a graft for transplantation based on the value(s) of cell-free nucleic acids (e.g. cell-free DNA) obtained.
  • assessing the suitability comprises providing a determination of the suitability of the graft for transplantation. The determination may be binary (e.g. suitable or not suitable). For example, the suitability of a graft for transplantation may be assessed based upon the value of short-fragment cell-free DNA obtained.
  • any one of the methods provided herein comprises correlating an increase in one or more values (e.g., for an amount of total and/or specific cell-free nucleic acids (such as DNA)) with unsuitability or declining suitability or a decrease in one or more values (e.g., for an amount of total and/or specific cell-free nucleic acids (such as DNA)) with suitability or increasing suitability.
  • an increased value of short-fragment cell- free DNA e.g. total and/or specific short fragment cell-free DNA
  • a decrease in short-fragment cell-free DNA (e.g. total and/or specific short-fragment cell-free DNA) in a sample is correlated with suitability or increasing suitability of a graft for transplantation.
  • the amount of long DNA fragments, short DNA fragments, and the amount of total and/or specific short fragment cell-free DNA is obtained and used to assess the suitability of a graft for transplantation.
  • the ratio of long: short DNA fragments (or short: long DNA fragments) is obtained and the amount of total and/or specific short fragment cell-free DNA is obtained, and the combination of these values is used to assess suitability of a graft for transplantation into a subject.
  • an increased ratio of long: short fragment DNA and an increased amount of total and/or specific short fragment cell-free DNA indicates unsuitability or diminished suitability of a graft for transplantation.
  • correlating comprises comparing a level (e.g., concentration, ratio or percentage) to a threshold value or value from another point in time to determine suitability, or increasing or decreasing suitability.
  • a level e.g., concentration, ratio or percentage
  • Any one of the methods provided herein can include one or more steps of comparing the values for an amount of nucleic acids (such as DNA) to a threshold value or a value from a different point in time to assess the suitability of the graph.
  • the method may further include additional test(s) for assessing suitability of the graph. The additional test(s) may employ any one of the methods provided herein or methods known in the art.
  • the reports include one or more threshold values. Reports may be provided in oral, written (or hard copy) or electronic form, such as in a form that can be visualized or displayed. In some embodiments, the “raw” results for each assay as provided herein are provided in a report, and from this report, further steps can be taken to analyze the amount(s) nucleic acids (such as DNA). In other embodiments, the report provides multiple values for the amounts of nucleic acids (such as DNA).
  • a clinician may assess the suitability of a graft for transplantation or the need to monitor the graft over time or treatment or some other remedial action.
  • the methods comprise transplanting the graph or starting, ending, or modifying treatment based on the results.
  • the amounts are in or entered into a database.
  • a database with such values is provided. From the amount(s), a clinician may assess the need for a treatment or monitoring. Accordingly, in any one of the methods provided herein, the method can include assessing the amount(s) at more than one point in time. Such assessing can be performed with any one of the methods or compositions provided herein.
  • the method can include assessing the amount of nucleic acids (such as DNA) at another point in time or times. Such assessing can be performed with any one of the methods provided herein.
  • nucleic acids such as DNA
  • Methods for determining total cell-free nucleic acids (such as DNA) as well as specific cell-free nucleic acids (such as DNA) are provided herein or are otherwise known in the art.
  • the methods of PCT Application No. PCT/US2016/030313, herein incorporated by reference in its entirety may be used for determining a value for the amount of specific cell-free nucleic acids (such as DNA) in a sample as provided herein.
  • any one of the methods provided herein may include the steps of any one of the methods described in PCT Application No. PCT/US2016/030313.
  • US-2015-0086477-A1 are also incorporated herein by reference and such methods can be included as part of any one of the methods provided herein for determining a value for the amount of cell-free nucleic acids (such as DNA).
  • amplification with PCR such as real-time PCR or digital PCR, may be used to determine a value for the amount of total cell-free nucleic acids (such as DNA) and/or specific cell-free nucleic acids (such as DNA).
  • the quantification is obtained for each target relative to a standard, such as an internal standard, that is spiked into a sample(s).
  • the total cell-free nucleic acids (such as DNA) is determined with Taqman Real-time PCR using RNase P as a target.
  • Other methods are provided elsewhere herein or would be apparent to those of ordinary skill in the art. Any one of the methods provided herein, can include any one of the methods of determining a value provided herein.
  • any one of the methods provided herein may include steps of a quantitative assay that makes use of mismatch amplification (e.g., MOMA) in order to determine a value for an amount of specific cell-free nucleic acids (such as DNA).
  • a quantitative assay that makes use of mismatch amplification (e.g., MOMA) in order to determine a value for an amount of specific cell-free nucleic acids (such as DNA).
  • the method further comprises selecting informative results of the amplification-based quantification assays, such as PCR quantification assays.
  • the selected informative results are averaged, such as a median average.
  • the results can be further analyzed with Robust Statistics.
  • the results can be further analyzed with a Standard Deviation, such as a Robust Standard Deviation, and/or Coefficient of Variation, such as a Robust Coefficient of Variation, or % Coefficient of Variation, such as a % Robust Coefficient of Variation.
  • the informative results of the amplification-based quantification assays are selected based on the genotype of the non-specific nucleic acids and/or specific nucleic acids.
  • the method further comprises obtaining the genotype of the non-specific nucleic acids and/or specific nucleic acids.
  • the plurality of SNV targets is at least 45, 48, 50, 55, 60, 65, 70, 75, 80, 85 or 90 or more. In some embodiments of any one of such mismatch methods, the plurality of SNV targets is at least 90, 95 or more targets. In some embodiments of any one of such mismatch methods, the plurality of SNV targets is less than 90, 95 or more targets. In some embodiments of any one of such mismatch methods, the plurality of SNV targets is less than 105 or 100 targets.
  • the mismatched primer(s) is/are the forward primer(s). In some embodiments of any one of such mismatch methods, the reverse primers for the primer pairs for each SNV target is the same.
  • Primers for use in such assays may be obtained, and any one of the methods provided herein can include a step of obtaining one or more primer pairs for performing the quantitative assays.
  • the primers possess unique properties that facilitate their use in quantifying amounts of nucleic acids.
  • a forward primer of a primer pair can be mismatched at a 3’ nucleotide (e.g., penultimate 3’ nucleotide).
  • this mismatch is at a 3’ nucleotide but adjacent to the SNV position.
  • the mismatch positioning of the primer relative to a SNV position is as shown in Fig. 1.
  • “Specific amplification” refers to the amplification of a specific target without substantial amplification of another nucleic acid or without amplification of another nucleic acid sequence above background or noise. In some embodiments, specific amplification results only in the amplification of the specific allele.
  • “single nucleotide variant” refers to a nucleic acid sequence within which there is sequence variability at a single nucleotide. In some embodiments, the SNV is a biallelic SNV, meaning that there is one major allele and one minor allele for the SNV. In some embodiments, the SNV may have more than two alleles, such as within a population.
  • a “minor allele” refers to an allele that is less frequent in a set of nucleic acids, for a locus, while a “major allele” refers to the more frequent allele in a set of nucleic acids.
  • the methods provided herein can quantify nucleic acids of major and minor alleles within a mixture of nucleic acids even when present at low levels, in some embodiments.
  • the nucleic acid sequence within which there is sequence identity variability is generally referred to as a “target”.
  • a “SNV target” refers to a nucleic acid sequence within which there is sequence variability at a single nucleotide.
  • the SNV target has more than one allele, and in preferred embodiments, the SNV target is biallelic.
  • the SNV target is a SNP target. In some of these embodiments, the SNP target is biallelic.
  • the amount of nucleic acids is determined by attempting amplification-based quantitative assays, such as quantitative PCR assays, with primers for a plurality of SNV targets.
  • a “plurality of SNV targets” refers to more than one SNV target where for each target there are at least two alleles.
  • each SNV target is expected to be biallelic and a primer pair specific to each allele of the SNV target is used to specifically amplify nucleic acids of each allele, where amplification occurs if the nucleic acid of the specific allele is present in the sample.
  • the mismatch primer is the forward primer.
  • the reverse primer of the two primer pairs for each SNV target is the same.
  • the forward and reverse primers are designed to bind opposite strands (e.g., a sense strand and an antisense strand) in order to amplify a fragment of a specific locus of the template.
  • the forward and reverse primers of a primer pair may be designed to amplify a nucleic acid fragment of any suitable size to detect the presence of, for example, an allele of a SNV target according to the disclosure.
  • Any one of the methods provided herein can include one or more steps for obtaining one or more primer pairs as described herein.
  • “informative results” as provided herein are the results that can be used to quantify the level of nucleic acids in a sample.
  • the amount of specific- and/or non-specific nucleic acids represents an average across informative results for the nucleic acids, respectively.
  • this average is given as an absolute amount or as a percentage.
  • this average is the median.
  • the amount, such as ratio or percentage, of specific nucleic acids may be determined with the quantities of the major and minor alleles as well as genotype, as needed.
  • the alleles can be determined based on prior genotyping (e.g., of the recipient or potential recipient and/or the subject from which a graft is obtained, respectively).
  • Methods for genotyping are well known in the art. Such methods include sequencing, such as next generation, hybridization, microarray, other separation technologies or PCR assays. Any one of the methods provided herein can include steps of obtaining such genotypes.
  • the primer pairs described herein may be used in a multiplex assays, such as multiplex PCR assays. Accordingly, in some embodiments, the primer pairs are designed to be compatible with other primer pairs in a PCR reaction. For example, the primer pairs may be designed to be compatible with at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, etc. other primer pairs in a PCR reaction. As used herein, primer pairs in a PCR reaction are “compatible” if they are capable of amplifying their target in the same PCR reaction.
  • primer pairs are compatible if the primer pairs are inhibited from amplifying their target nucleic acid (such as DNA) by no more than 1%, no more than 2%, no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 30%, no more than 35%, no more than 40%, no more than 45%, no more than 50%, or no more than 60% when multiplexed in the same PCR reaction.
  • Primer pairs may not be compatible for a number of reasons including, but not limited to, the formation of primer dimers and binding to off-target sites on a template that may interfere with another primer pair.
  • the primer pairs of the disclosure may be designed to prevent the formation of dimers with other primer pairs or limit the number of off-target binding sites.
  • Exemplary methods for designing primers for use in a multiplex assays are known in the art and are otherwise described herein.
  • the mismatch amplification-based quantitative assay is any quantitative assay whereby nucleic acids are amplified and the amounts of the nucleic acids can be determined.
  • Such assays include those whereby nucleic acids are amplified with the MOMA primers as described herein and quantified.
  • Such assays include simple amplification and detection, hybridization techniques, separation technologies, such as electrophoresis, next generation sequencing and the like.
  • the quantitative assays include quantitative PCR assays. Quantitative PCR include real-time PCR, digital PCR, TaqMan, etc.
  • the PCR is “Real-time PCR”.
  • PCR refers to a PCR reaction where the reaction kinetics can be monitored in the liquid phase while the amplification process is still proceeding.
  • real-time PCR offers the ability to simultaneously detect or quantify in an amplification reaction in real time. Based on the increase of the fluorescence intensity from a specific dye, the concentration of the target can be determined even before the amplification reaches its plateau.
  • the PCR may be digital PCR.
  • Digital PCR involves partitioning of diluted amplification products into a plurality of discrete test sites such that most of the discrete test sites comprise either zero or one amplification product.
  • the amplification products are then analyzed to provide a representation of the frequency of the selected genomic regions of interest in a sample. Analysis of one amplification product per discrete test site results in a binary “yes-or-no” result for each discrete test site, allowing the selected genomic regions of interest to be quantified and the relative frequency of the selected genomic regions of interest in relation to one another be determined.
  • multiple analyses may be performed using amplification products corresponding to genomic regions from predetermined regions.
  • Results from the analysis of two or more predetermined regions can be used to quantify and determine the relative frequency of the number of amplification products.
  • Using two or more predetermined regions to determine the frequency in a sample reduces a possibility of bias through, e.g., variations in amplification efficiency, which may not be readily apparent through a single detection assay.
  • Methods for quantifying DNA using digital PCR are known in the art and have been previously described, for example in U.S. Patent Publication number US20140242582, which is herein incorporated by reference in its entirety.
  • Any one of the methods provided herein can comprise extracting nucleic acids, such as cell-free DNA. Such extraction can be done using any method known in the art or as otherwise provided herein (see, e.g., Current Protocols in Molecular Biology, latest edition, or the QIAamp circulating nucleic acid kit or other appropriate commercially available kits).
  • An exemplary method for isolating cell-free DNA from blood is described.
  • Blood containing an anti-coagulant such as EDTA or DTA is collected.
  • the plasma, which contains cf-DNA is separated from cells present in the blood (e.g., by centrifugation or filtering).
  • An optional secondary separation may be performed to remove any remaining cells from the plasma (e.g., a second centrifugation or filtering step).
  • the cf-DNA can then be extracted using any method known in the art, e.g., using a commercial kit such as those produced by Qiagen.
  • a pre amplification step is performed.
  • An exemplary method of such a pre-amplification is as follows, and such a method can be included in any one of the methods provided herein.
  • Approximately 15 ng of cell-free plasma DNA is amplified in a PCR using Q5 DNA polymerase with approximately 13 targets where pooled primers were at 4uM total. Samples undergo approximately 25 cycles. Reactions are in 25 ul total.
  • samples can be cleaned up using several approaches including AMPURE bead cleanup, bead purification, or simply ExoSAP-ITTM, or Zymo.
  • kits may comprise components necessary, useful, or sufficient to perform one or more aspects of the methods described herein.
  • the kit may comprise any one or more of sample collection tubes, centrifuge tubes, sample storage tubes, packaging materials necessary for shipping the sample, materials necessary for isolating short-fragment DNA and/or removing long fragment DNA from the sample (e.g. beads, particles, resins, buffers, etc.) materials for quantifying cell-free DNA in the sample (e.g. primers, probes, buffers, etc.), devices and software for measuring cell-free DNA, and instructions for using the kit.
  • embodiments of the invention may be implemented as one or more methods, of which an example has been provided.
  • the acts performed as part of the method(s) may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different from illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.
  • the Toronto EVLP method utilized involves gradual re-warming of the lungs to normal core body temperature in conjunction with gradual increase in vascular flow, targeting a perfusion flow of 40% donor-predicted cardiac output (CO). It utilizes protective lung ventilation and an acellular perfusate with increased colloid osmotic pressure achieved through inclusion of human serum albumin and Dextran 40. This widely applied methodology is FDA approved under a humanitarian device exemption (HOE).
  • HOE humanitarian device exemption
  • STEENTM solution is a buffered extracellular solution that includes human serum albumin (HSA) to provide optimal osmotic pressure, facilitated by Dextran 40, a mild scavenger to coat and protect the endothelium from excessive leucocyte interaction.
  • HSA human serum albumin
  • STEENTM solution is designed to facilitate prolonged evaluation of lung transplantation options and to and promote health of isolated lungs ex vivo.
  • EVLP using STEENTM solution thus has the potential to be able to increase the likelihood that previously rejected , but ex vivo rehabilitated lungs could be used to increase the availability of potential organs for lung transplantation.
  • Dextran 40 (MW 40,000) was solubilized in H20 at weight to weight concentrations of 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56% and 0.78%.
  • Table 1 Reference gene qPCR measuring detectable gDNA in H20 supplemented with 0.78-50% Dextran 40.
  • Procedure and Results One ml volumes of STEEN solutionTM supplemented with 0 or 1.5 IU/ml heparin were spiked with gDNA sheared to approximate cfDNA size (average 150 bp). Spiked samples were measured directly without extraction for total DNA levels using reference gene qPCR. Results are shown in FIG. 1.
  • the cfDNA concentration in the EVLP perfusate solution should be quantified rapidly to be useful for clinical decision making. To meet this need for speed, it is important to develop a rapid DNA assay that does not include traditional extraction of the DNA measurement independent of DNA extraction. This data suggests that DNA can be measured by qPCR from STEEN SolutionTM without need to undergo a time-consuming extraction step.
  • Example 2 To better understand the findings of Example 2, we evaluated the ability to detect spiked sheared gDNA in STEEN containing heparin concentrations equivalent to pre- and post-replenishment of the EVLP circuit.
  • Heparin is a highly sulfonated glycosaminoglycan with the ability to bind a wide range of biomolecules including: DNA binding proteins such as initiation factors, elongation factors, restriction endonucleases, DNA ligase, DNA, and RNA polymerases. Because heparin is negatively charged like the phosphodiester bridges of DNA, it is possible that heparin inhibits the qPCR reaction by binding directly to DNA polymerase. If this is true, a bead-based DNA purification step to directly bind DNA and allow removal of background heparin would increase the percentage of detectable spiked gDNA (Example 4).
  • Procedure and Results STEEN Solution supplemented with 0, 1, 5, or 100 IU/ml heparin was spiked with 500 ng/ml of sheared gDNA. The DNA from these samples was then purified. Purification can be done using various methods, including bead purification. In this example, purification was done using AMPureXP beads and eluted in nuclease free water. The purified DNA was measured directly for total DNA levels using a reference gene qPCR method. Results are shown in FIG. 3.
  • IU/ml heparin was spiked with 25,000 copies of short DNA fragment control, which closely resembles cfDNA.
  • the DNA from these samples was then purified using AMPureXP beads and eluted in nuclease free water. The percentage of detectable short DNA fragment control was measured directly after purification using a reference gene qPCR method. Results are shown in Fig. 4.
  • Procedure and Results Two ml volumes of STEEN solution and plasma were spiked with sheared gDNA and incubated at both room temperature and 37°C for 0, 1, 3, 12, and 24 hours. DNA was then extracted using an automated DNA extraction workflow.
  • Table 2 Reference gene qPCR measuring total cfDNA concentration in EVLP clinical perfusate samples collected at hours 1, 2, 3, and a “0 hour” STEEN solution control.
  • Table 3 qPCR measuring the percent recovery of spiked short fragment DNA control from clinical EVLP perfusates at hours 1, 2, 3, and a “0 hour” STEEN solution control.
  • Table 5 Reference gene qPCR measuring DNA in EVLP clinical perfusate samples after size-selected purification.
  • Elevated long-fragment cfDNA ratios appear to be a characteristic of EVLP perfusates, unlike in vivo blood collections in which apoptotic fragments dominate. Regardless, the overall total concentration of cfDNA in clinical EVLP circuits, coupled with the rate of accumulative increase in that level over time, is a valuable indicator of lung health during EVLP. In addition, using DNA fragment size selective analyses demonstrated to be feasible: correlation of short vs long cfDNA fragment lengths with EVLP lung health is shown to be practical in EVLP circuit collections.
  • Embodiment 1 A method of assessing the suitability of a potential graft for transplantation, comprising: a) obtaining an amount of total short fragment cf-DNA and/or graft- specific short-fragment cf-DNA released from a graft prior to contacting the graft with blood cells from a potential recipient, and b) assessing the amount(s) to determine the suitability of the graft for transplantation.
  • Embodiment 2 The method of Embodiment 1, wherein obtaining the amount comprises performing a method to quantify the amount of total short fragment cf- DNA and/or graft- specific short fragment cf-DNA released by the graft.
  • Embodiment 3 The method of Embodiment 1 or 2, wherein the method further comprises performing a method to quantify the amount of total short fragment cf- DNA and/or graft- specific short-fragment cf-DNA released by the graft subsequent to contacting the graft or cells thereof with blood cells from a potential recipient.
  • Embodiment 4 A method of assessing the suitability of a graft for transplantation, comprising:
  • Embodiment 5 The method of Embodiment 4, wherein obtaining the amount comprises performing a method to quantify the amount(s) of total short fragment cell- free DNA and/or graft- specific short fragment cf-DNA in order to obtain the amount.
  • Embodiment 6 The method of any one of Embodiments 1-5, wherein the method further comprises determining an amount of cell lysis.
  • Embodiment 7 The method of Embodiment 6, wherein the determining the amount of cell lysis comprises performing fragment analysis.
  • Embodiment 8 The method of Embodiment 7, wherein the fragment analysis comprising quantifying long and/or short fragments.
  • Embodiment 9 The method of any one of the preceding Embodiments, wherein the method further comprises one, two, three or more centrifugation steps of the sample(s) prior to obtaining the amount of total short fragment cf-DNA and/or graft- specific short-fragment cf-DNA and/or cell lysis.
  • Embodiment 10 The method of any one of the preceding Embodiments, wherein the method further comprises obtaining the potential graft or cells thereof.
  • Embodiment 11 The method of any one of the preceding Embodiments, wherein the method further comprises obtaining an amount of total short fragment cf-DNA and/or graft- specific short-fragment cf-DNA and/or determining the amount of cell lysis at one or more additional time points.
  • Embodiment 12 The method of any one of the preceding Embodiments, wherein the method further comprises comparing the amount(s) of the total short fragment cf- DNA and/or graft- specific cf-DNA and/or amount of cell lysis with threshold value(s) or amount(s) obtained from additional time point(s).
  • Embodiment 13 The method of any one of the preceding Emsbodiment, wherein the amount(s) of the total short fragment cf-DNA and/or graft- specific cf-DNA and/or amount of cell lysis are obtained in a sample of media in which the potential graft or cells therefrom are contained or are in contact with.
  • Embodiment 14 The method of Embodiment 13, wherein the media is a storage, perfusion, or preservation media in which the graft is contained or in contact with.
  • Embodiment 15 The method of Embodiment 13 or 14, wherein the cf-DNA is released from the graft into the media.
  • Embodiment 16 The method of any one of the preceding Embodiments, wherein the amount(s) is/are obtained in a perfusate sample.
  • Embodiment 17 The method of any one of the preceding Embodiments, wherein the amount(s) is/are obtained in an EVLP sample.
  • Embodiment 18 The method of any one of the preceding Embodiments, wherein the method further comprises obtaining the blood cells (e.g., blood) from the potential recipient.
  • the blood cells e.g., blood
  • Embodiment 19 The method of any one of the preceding Embodiments wherein the potential graft or cells thereof are from a potential donor of the same species as the potential recipient.
  • Embodiment 20 The method of any one of the preceding Embodiments, wherein the potential graft is an organ or organs.
  • Embodiment 21 The method of Embodiment 20, wherein the organ or organs comprise a heart, a lung, or a heart and a lung.
  • Embodiment 22 The method any one of the preceding Embodiments, wherein the method further comprises providing the values for the amount(s) of total cell-free DNA and/or graft- specific cf-DNA and/or cell lysis in a report.
  • Embodiment 23 The method of any one of the preceding Embodiments, wherein the method further comprises providing a determination about the suitability of the graft.
  • Embodiment 24 The method of any one of the preceding Embodiments, wherein the graft is monitored over time.
  • Embodiment 25 The method of Embodiment 24, wherein the graft is assessed every 15 minutes, every 30 minutes, hourly, daily, weekly, bimonthly or monthly prior to transplantation.
  • Embodiment 26 The method of any one of the preceding Embodiments, wherein the total short fragment cell-free DNA and/or graft- specific short fragment cf-DNA and/or cell lysis is measured using a quantitative amplification method.
  • Embodiment 27 The method of Embodiment 26, wherein the quantitation amplification method is selected from real-time PCR, digital PCR, and mismatch PCR.
  • Embodiment 28 The method of any one of Embodiments 1-25, wherein the amount of total short fragment cell-free DNA and/or graft- specific short fragment cf-DNA and/or cell lysis is measured using next-generation sequencing.
  • Embodiment 29 The method of any one of the preceding Embodiments, wherein when the value for the amount(s) are above a threshold value or a value from a prior point in time, decreasing suitability of the graft or unsuitability of the graft for transplantation is indicated.
  • Embodiment 30 The method of any one of the preceding Embodiments, wherein when the value for the amount(s) are below a threshold value or a value from a prior point in time, increasing suitability of the graft or suitability of the graft for transplantation is indicated.
  • Embodiment 31 The method of any one of the preceding Embodiments, further comprising performing treatment of the graft, potential donor, and/or recipient, or providing information regarding such a treatment.
  • Embodiment 32 A report comprising the value(s) or amount(s) of total short fragment cell-free DNA, specific short fragment cell-free DNA, and/or cell lysis obtained by the method of any one of the preceding Embodiments.

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Abstract

La présente invention concerne des procédés et des compositions d'évaluation de la compatibilité d'une greffe pour la transplantation en mesurant les acides nucléiques totaux et/ou spécifiques sans cellules (tels que l'ADNac) et/ou la lyse cellulaire. Plus particulièrement, le procédé comprend l'obtention d'une quantité de fragment court d'ADNac total et/ou de fragment court d'ADNac spécifique d'une greffe potentielle (par exemple, ex vivo), par exemple, avant la mise en contact de la greffe potentielle avec les cellules sanguines d'un bénéficiaire potentiel, et/ou suite à la mise en contact de la greffe potentielle ou de ses cellules avec des cellules sanguines d'un bénéficiaire potentiel, et l'évaluation de la(des) quantité(s) pour déterminer la compatibilité de la greffe potentielle pour la transplantation.
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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11286530B2 (en) 2010-05-18 2022-03-29 Natera, Inc. Methods for simultaneous amplification of target loci
US11306359B2 (en) 2005-11-26 2022-04-19 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US11306357B2 (en) 2010-05-18 2022-04-19 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11322224B2 (en) 2010-05-18 2022-05-03 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11319596B2 (en) 2014-04-21 2022-05-03 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11326208B2 (en) 2010-05-18 2022-05-10 Natera, Inc. Methods for nested PCR amplification of cell-free DNA
US11332785B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11332793B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for simultaneous amplification of target loci
US11339429B2 (en) 2010-05-18 2022-05-24 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11390916B2 (en) 2014-04-21 2022-07-19 Natera, Inc. Methods for simultaneous amplification of target loci
US11479812B2 (en) 2015-05-11 2022-10-25 Natera, Inc. Methods and compositions for determining ploidy
US11485996B2 (en) 2016-10-04 2022-11-01 Natera, Inc. Methods for characterizing copy number variation using proximity-litigation sequencing
US11519035B2 (en) 2010-05-18 2022-12-06 Natera, Inc. Methods for simultaneous amplification of target loci
US11519028B2 (en) 2016-12-07 2022-12-06 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US11525159B2 (en) 2018-07-03 2022-12-13 Natera, Inc. Methods for detection of donor-derived cell-free DNA
US11746376B2 (en) 2010-05-18 2023-09-05 Natera, Inc. Methods for amplification of cell-free DNA using ligated adaptors and universal and inner target-specific primers for multiplexed nested PCR
US11931674B2 (en) 2019-04-04 2024-03-19 Natera, Inc. Materials and methods for processing blood samples
US11939634B2 (en) 2010-05-18 2024-03-26 Natera, Inc. Methods for simultaneous amplification of target loci

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019118926A1 (fr) * 2017-12-14 2019-06-20 Tai Diagnostics, Inc. Évaluation de la compatibilité d'une greffe pour la transplantation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019118926A1 (fr) * 2017-12-14 2019-06-20 Tai Diagnostics, Inc. Évaluation de la compatibilité d'une greffe pour la transplantation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AGBOR-ENOH ET AL.: "Donor-derived cell -free DNA predicts allograft failure and mortality after lung transplantation", EBIOMEDICINE, vol. 40, 26 January 2019 (2019-01-26), pages 541 - 553, XP055807957 *
KANOU ET AL.: "Cell -Free DNA in Human Ex-Vivo Lung Perfusate as a Potential Biomarker to Predict the Risk of Primary Graft Dysfunction (PGD) in Lung Transplantation", THE JOURNAL OF HEART AND LUNG TRANSPLANTATION, vol. 36, no. 45, 2017, pages 187 *

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11306359B2 (en) 2005-11-26 2022-04-19 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US11746376B2 (en) 2010-05-18 2023-09-05 Natera, Inc. Methods for amplification of cell-free DNA using ligated adaptors and universal and inner target-specific primers for multiplexed nested PCR
US11306357B2 (en) 2010-05-18 2022-04-19 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11312996B2 (en) 2010-05-18 2022-04-26 Natera, Inc. Methods for simultaneous amplification of target loci
US11322224B2 (en) 2010-05-18 2022-05-03 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11939634B2 (en) 2010-05-18 2024-03-26 Natera, Inc. Methods for simultaneous amplification of target loci
US11286530B2 (en) 2010-05-18 2022-03-29 Natera, Inc. Methods for simultaneous amplification of target loci
US11326208B2 (en) 2010-05-18 2022-05-10 Natera, Inc. Methods for nested PCR amplification of cell-free DNA
US11332785B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11332793B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for simultaneous amplification of target loci
US11339429B2 (en) 2010-05-18 2022-05-24 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11525162B2 (en) 2010-05-18 2022-12-13 Natera, Inc. Methods for simultaneous amplification of target loci
US11519035B2 (en) 2010-05-18 2022-12-06 Natera, Inc. Methods for simultaneous amplification of target loci
US11482300B2 (en) 2010-05-18 2022-10-25 Natera, Inc. Methods for preparing a DNA fraction from a biological sample for analyzing genotypes of cell-free DNA
US11414709B2 (en) 2014-04-21 2022-08-16 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11530454B2 (en) 2014-04-21 2022-12-20 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11486008B2 (en) 2014-04-21 2022-11-01 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11408037B2 (en) 2014-04-21 2022-08-09 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11390916B2 (en) 2014-04-21 2022-07-19 Natera, Inc. Methods for simultaneous amplification of target loci
US11319596B2 (en) 2014-04-21 2022-05-03 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11371100B2 (en) 2014-04-21 2022-06-28 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11319595B2 (en) 2014-04-21 2022-05-03 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11479812B2 (en) 2015-05-11 2022-10-25 Natera, Inc. Methods and compositions for determining ploidy
US11946101B2 (en) 2015-05-11 2024-04-02 Natera, Inc. Methods and compositions for determining ploidy
US11485996B2 (en) 2016-10-04 2022-11-01 Natera, Inc. Methods for characterizing copy number variation using proximity-litigation sequencing
US11530442B2 (en) 2016-12-07 2022-12-20 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US11519028B2 (en) 2016-12-07 2022-12-06 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US11525159B2 (en) 2018-07-03 2022-12-13 Natera, Inc. Methods for detection of donor-derived cell-free DNA
US11931674B2 (en) 2019-04-04 2024-03-19 Natera, Inc. Materials and methods for processing blood samples

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